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Home , Cambarus, Cambarus aculabrum, Cambarus cryptodytes, Cambarus zophonastes, Neosho River

W 2800.7 E56s No.E-5 1989/90 c.3

SECTION 6 ENDANGERED SPECIES ACT

OKLAHOMA o

FEDERAL AID PROJECT E-5 SURVEY AND SPECIES DETERMINATION OF CAVE IN JUNE 1, 1989 - MAY 31, 1990 This study was undertaken to determine the identification, distribution and abundance of troglobitic crayfish in the Ozark Plateau region of Oklahoma. Specific objectives were:

1. To survey sites with historical records of cave crayfish in the Ozarks of Oklahoma, and determine the current status of cave crayfish at those sites.

2. To survey other potential sites for cave crayfish for which no records of occurrence exist.

3. To determine which species are in which caves using tissue electrophoresis to clarify genetic similarity of populations.

Information from this study is expected to be useful in making decisions on listing and protection needs for cave crayfish in the Ozark Plateau of Oklahoma, and .

Cave crayfish are troglobitic, entirely dependent upon a subterranean existence. While a divergent group taxonomically, they possess convergent characteristics of deceased pigmentation and claw musculature. They are represented in the Americas by species in eight families, which also have epigean species (Hobbs et. al. 1977). Four species have been named from the western part of the Ozark Plateau, which includes Oklahoma, Arkansas and Missouri. All are members of the subgenus Jugicambarus in the (Hobbs and Brown 1987). These are considered to have derived from an epigean parental stock in the southern Appalachian Mountains (Hobbs et. al. 1977, Hobbs and Barr 1960). The only troglobitic species from the subgenus Jugicambarus that is known to occur outside of the Ozark Plateau is from the panhandle of northern Florida (Hobbs and Barr 1960). The species Cambarus hubricti is a troglobitic species of a different subgenus that occurs in the eastern part of the Ozark Plateau (Gardner 1986, Hobbs et. al. 1977, Hobbs and Barr 1960).None of the troglobitic species from the Ozarks are known to co-occur.

The main morphological feature used to distinguish the troglobitic species of Jugicambarus is the gonopod (first pleopod) of males in reproductive condition, called first form males (in this report abbreviated Male-I). While non-sexual characters can be used for tentative identifications, confirmed identifications must be based on the gonopods of Male-Is (Hobbs et. al. 1977, Hobbs 1976 and personal communication). The status of cave crayfish in Oklahoma is particularly problematic, principaUy because adequate data have not been coUected. Of the four species of the subgenus Jugicambarus described from the Ozark Plateau, two are thought to occur in Oklahoma. Cambarus setosus Faxon is known from eight counties in southwestern Missouri (Figg and Lister 1990). Prior to the mid 1970s, it was thought that C. setosus probably occurred in Oklahoma (Black 1971, Creaser and Ortenburger 1933, Williams 1954). On the basis of specimens that were not Male-I from Twin Cave and "Spring Creek, 5 mi. S. of Locust Grove" in Oklahoma, Hobbs and Barr (1960) tentatively caUed populations from those caves C. setosus. Cambarus tartarus Hobbs and Cooper was described from type specimens from a single cave in Oklahoma (Hobbs and Cooper 1972). Without having specimens to examine from three other Oklahoma caves (Jail, Rodman, and Star), the cave crayfish from those caves were tentatively assigned to C. tartarus (Hobbs et. al. 1977). Hobbs and Brown has been described from two caves in Arkansas, both of which are close to the Oklahoma state line (Hobbs and Brown 1987). Hobbs and Bedinger is known from a single cave in Stone County, Arkansas (Hobbs and Bedinger 1964), considerably to the east of Oklahoma and aU"other known sites for species of Jugicambarus (Figg and Lister 1990, Hobbs and Brown 1987, Hobbs et. al 1977).

Prior to this study, cave surveys in the Ozark Plateau of Oklahoma had not focused on status assessment of cave crayfish, so records have been scattered and incomplete.

Cambarus setosus: none Cambarus tartarus: Candidate 2, petitioned in 1986 for listing as endangered (Brown 1986). Candidate 2, petitioned for listing as endangered. Endangered, Federal Register for April 7, 1987.

As troglobites, cave crayfish are obligate inhabitants of subterranean aquatic systems. In the Ozark Plateau of Oklahoma, some caves have sizeable perennial streams that follow the main course of the cave for its length; some caves have perennial streams and/or pools in only a part of the cave; and some caves have ephemeral streams and/or pools that have water only when water levels are raised by rains. Based on a few records on microhabitat use in Oklahoma caves, it appears that the cave crayfish are found more often in pools and lower gradient parts of streams. In all aquatic caves, water levels and gradient fluctuate with flood conditions. In most caves and subterranean aquatic systems, the lack of primary producers results in dependency of primary consumers and detritivores on the import of organic material from surface areas. Thus, though caves in Oklahoma may not have cave crayfish at all times, they may be important habitat with respect to sources for nutrient inputs.

The study area was in that portion of the Ozark Plateau of Oklahoma in which aquatic caves are known to occur. This includes parts of Delaware, Mayes and Ottawa counties, principally where the surface geology is limestone and chert (Figures 1 and 2).

Personal communication with Dr. Arthur Brown, Dr. Jeffery Black and Mr. William Puckette, who have seen cave crayfish in Oklahoma caves, indicated that all known specimens deposited in a museum are in the collection of the U. S. National Museum. I visited that collection in March of 1990 and examined all specimens of cave crayfish from Oklahoma. In addition, published and unpublished records were obtained and reviewed. Historical records also were obtained for the Ozark cavefish, Amblyopsis rosae, on the assumption that habitat suitable for a troglobitic fish might be suitable for a troglobitic crayfish.

Caves selected for field survey work included those with reported records of cave crayfish and cavefish and those aquatic caves in close proximity to caves with historical records. The rationale was that caves in proximity were most likely to be in the same hydrological system as caves with historical records. Aquatic caves with no records of cave crayfish or cavefish were not visited if they had had frequent checks by W. L. Puckette during winter bat surveys.

Figure 2. Detail of region in Oklahoma with aquatic solution caves limestone (shaded area).

CHEROKEE surveys were conducted with W. L. Puckette who is most familiar with caves in the Ozarks of Oklahoma. Accessible ephemeral and perennial aquatic areas of each cave were visited. Two caves were visited two times because water levels were extremely low during the first visit and did not afford adequate assessment of crayfish use of those caves. A third cave was revisited to collect a male for morphometric examination.

Electrophoretic Analysis of Tissue Proteins:

When cave crayfish were found in a cave, a claw (usually the left claw) was removed from individuals that possessed two claws if the individuals appeared healthy. Samples were sent on dry ice to the Missouri Department of Conservation for electrophoretic analysis of the claw muscle tissue. Dr. Jeffery Koppelman performed the analyses along with samples of cave crayfish from Ozark Plateau caves in Missouri. We were unsuccessful in obtaining samples from Arkansas caves, principally because the people in that state who are familiar with Ozark caves were not available to do the work.

Because of the sensitivity of caves of the Ozark Plateau and extreme pressures for human visitation use, the precise location of caves we visited is not presented in this report. Instead, by agreement with the U. S. Fish & Wildlife Service (Steven Hensley, Ecological Services Office, Tulsa, Oklahoma, personal communication), the location of known caves is given to the nearest four sections in Appendix 1. Caves are identified by the same code system that has been used in studies of endangered bats in caves in the study area.

A total of 16 caves were located and surveyed, one spring was visited, and we were unable to locate one cave for which an historical record exists. A summary of the results of both the historical record and field surveys is given in Table 1. The results are detailed in Appendix 1. Cave crayfish were found in four caves, coded as DL-38, DL-39, DL-74 and DL-91 (Table 2 and Appendix I). Morphological measurements taken on live individuals were not an adequate means of distinguishing the species (Table 2 and H. H. Hobbs, Jr., personal communication). Males in reproductive condition (Male-I) were distinguished from non-reproductive males (Male-II) and examined for species determination. One Male-I was captured, examined and released in each of caves DL- 38, DL-74, and DL-91. Although the gonopods were viewed under a lOX hand lens, a positive determination of species could not be made. On the basis of published descriptions, Cambarus setosus can be distinguished from C. tartarus by a notch on the distal end of the central projection of the gonopod; the notch is lacking in C. setosus. No notch was seen on the gonopod of the Male-Is from DL-38, DL-74 or DL-91. Because of the apparent low density of these cave populations, no voucher specimens Summary of historical records and results of 1989-90 field surveys for cave crayfish in the Ozark Plateau of Oklahoma.

Pre-study No. of Cave Site Record record of crayftsh seen per Probability code no. of caveftsh? cave crayfISh? visit in this study? cave crayfish occur

DL-91 1 yes yes 13 occur DL-38 2 yes yes 4 occur DL-21 3 yes no 0 not probable DL-22 4 no no 0 not probable DL-I46 5 no no 0 not probable DL-39 6 no yes 6 occur DL-49 sump 7 no no 0 not probable DL-49 #1 8 no no?* 0 possible intermittent use DL-49 #2 9 yes no?* 0 possible intermittent use DL-49 #3 10 no yes 0 probable intermittent use DL~84 11 no no 0 not probable DL-74 12 no yes 17 occur DL-16 13 no no 0 not probable DL-64 14 no yes 0 possible intermittent use DL-61 15 no no 0 not probable OT-19 16 yes no 0 unknown Spring Creek no yes not found historical site Locust Grove Spring 17 no no** 0 possibly flushed out from spnng DL-19 no no?* not surveyed not probable

* Looney (1975) reported cave crayftsh from this cave, but without documentation. ** J.H. Black (personal communication) suggested that this may be the actual site reported as Spring Ck. Measurements (mm) of Cave Crayfish (Cambarus spp.) captured and released in Oklahoma caves.

Site Indiv. Areola Carapace Chelae Cave No. No. Sex L W L W L W

DL-91 1 1 F 11.2 3.2 25.1 10.5 11.3 5.5 2 F 10.1 2.8 19.5 9.4 8.1 4.2 3 F 8.0 1.3 15.4 6.4 6.0 2.3 4 M-II 9.4 2.5 17.2 8.1 6.0 3.3 5 F 8.6 1.2 16.3 7.1 6.0 2.9 6 F 7.4 1.4 14.0 6.0 7 M-I 7.8 2.1 16.3 9.4 7.4 3.8 8 F 10.2 2.2 20.5 8.0 6.0 4.6 9 F 10.4 3.6 15.6 7.9 5.7 2.8 10 M-II 9.1 1.2 18.1 6.9 4.7 2.6 11 F 9.5 2.0 18.3 6.9 5.3 1.5

DL-38 2 1 M-I 5.2 1.0 11.5 5.2 4.5 2.4 2 F 7.0 1.3 12.1 5.5 3.9 1.5 3 M-II 8.5 1.6 16.5 6.9 6.0 3.4

DL-74 12 1 F 8.2 1.1 21.2 9.1 2 F 8.5 1.6 21.8 9.0 8.1 4.6 3 M-II 8.3 1.3 17.0 7.0 6.2 3.0 4 F 8.3 1.3 17.7 7.4 6.2 3.2 5 F 5.9 .9 13.6 4.9 5.3 2.6 6 M-II 5.1 .9 11.3 4.2 4.1 1.4 7 F 11.6 1.4 23.4 11.3 11.0 7.2 8 F 7.7 1.2 16.5 7.5 5.7 2.8 9 F 11.9 3.0 24.6 10.6 10.1 4.8 10 M-I 8.0 1.4 18.6 7.3 4.9 2.2 11 M-II 6.7 1.1 14.1 6.4 3.4 1.2

DL-39 6 1 F 11.1 1.5 23.9 10.5 8.5 5.3 2 F 11.5 1.6 24.7 11.9 9.2 6.1 3 F 11.5 .9 23.5 10.4 9.4 5.1 4 M-II 8.5 .6 18.7 8.0 6.8 3.4 5 F 9.5 1.3 19.7 9.2 7.3 4.3 6 F 10.1 .9 19.4 8.2 6.9 3.7 were collected in this study. On the basis of the gonopods of two Male-I individuals from DL-91 (D. S. National Museum # 133842 & 148793), H. H. Hobbs, Jr. assigned this species to C. setosus. No Male-I vouchers exist for caves DL-38 or DL-74. A second visit was made to DL-38 to obtain a voucher. No Male-I was captured, but a Male-II was taken and is being held alive until it molts to Male-I condition. Cave crayfish from those three caves had more robust chelae than the cave crayfish from DL-39. Though no Male-I was found in DL-39 during our field surveys, the species from that cave has been confirmed by H. H. Hobbs, Jr. to be C. tartarus, based on type specimens in the D. S. National Museum (see Appendix I).

Crayfish were not found during either of two visits to caves DL-49 #3 and DL-64, though historical records of occurrence exist for both. In each of those caves, human access to water is limited, especially during dry weather (such as we had in the fall of 1989) when water levels are low. Those caves and their associated subterranean streams probably are used by cave crayfish, but detection of crayfish presence is limited by access to water. In both caves, there was evidence of extensive flooding with mud and debris washed into them during the spring rains of 1990. For this reason, DL-49 #3 may be an important nutrient input source for the cave crayfish. However, cave DL-64 is at the upstream end of a series of resurgences in the stream. It appears most likely that troglobites would be restricted to subterranean areas upstream from the cave, while nutrients flushed into the cave would be transported downstream. The historic crayfish record from DL-64 may represent an individual being flushed out of its usual habitat upstream. Cave DL-19 was mentioned in a popular article by Looney (1975) as having had a report of a cave crayfish, but we found no such reports and no specimens. W. L. Puckette (personal communication) has visited the cave three times and not found troglobites present. The cave does not appear to be good habitat because of its tendency to flush (Appendix I).

Attempts to locate an historical cave site, Spring Creek 5 mi. south of Locust Grove in Mayes County, were unsuccessful. Four long-time landowners along Spring Creek had not heard of a cave in that area, and a search of cliffs along the bank did not reveal any. The specimen collected from the site (D. S. National Museum, H. H. Hobbs, Jr. # 5-2150-1) was an immature female, which H. H. Hobbs, Jr. tentatively assigned to C. setosus (Hobbs et. al. 1977, personal communication). Because that area is disjunct from all other caves in Oklahoma that have confirmed or reported occurrences of cave crayfish, it would be most interesting to learn the identity of the cave crayfish from that cave.

The results of the electrophoretic analyses are given in a report by Koppelman (1990, Appendix II). Caves DL-38, DI-74 and DL-91 (Oklahoma sites 1, 2 and 12 in the report) show close genetic relatedness. While electrophoretically they are most similar to C. aculabrum, their similarity distance from it is quite large (0.201). Morphometrically, the Male-I gonopod is more similar to C. setosus. Electrophoretically, C. tartarus was not distinguishable from C. setosus from southern .Missouri, though morphologically they can be readily distinguished on the basis of Male- I gonopods.

All of the caves visited are in the Neosho (Grand Lake) drainage. Delaware County caves with cave crayfish records are in relatively close proximity to one another. Caves DL-74 and DL-91 are in the same surface tributary drainage to Grand Lake and close to the surface tributary in which DL-38 is located. Caves DL-39 and DL-49 are in nearby small drainages that are tributaries to a larger tributary to Grand Lake, and DL-64 is in a more distant tributary to that same larger tributary. Spring Creek, 5 mi. south of Locust Grove flows into Grand Lake farther south. Based on proximity of caves and surface waters, cave crayfish in DL-38, DL-74 and DL-91 would be most likely to be closely related. Crayfish in caves DL-39, DL-49 and probably DL-64 would be expected to be more closely related to one another than 10 other populations, and the Spring Creek cave crayfish population is most isolated.

Cambarus tartarus appears to be a distinct species, based on its distinct morphology Hobbs and Cooper 1972). Its high electrophoretic similarity to Cambarus setosus from southern Missouri indicates that it is recently derived from that species. Although DL- 39 is the only cave from which it has been confirmed, based on proximity of surface drainages, it is possible that the cave crayfish associated with DL-49 and DL-64 are C. tartarus as well.

Although C. aculabrum was not an original focus of this study, it is of interest because of the results of the electrophoretic analyses and the proximity of the species to Oklahoma. Cave crayfish from DL-38, DL-74 and DL-91 are genetically similar to one another, based on similarity distance of the tissue proteins. Cambarus aculabrum is their nearest relative, but with a rather large similarity distance (0.20). Those four samples (three from Delaware County plus C. aculabrum) then cluster with C. setosus from southern Missouri at only a slightly greater similarity distance (0.25). Based on its distinct morphology described in its species account (Hobbs and Brown 1987) and its genetic uniqueness revealed by Koppelman (1990), a species designation is entirely appropriate for C. aculabrum.

A final species determination for the three similar Delaware County cave crayfish populations (DL-38, DL-74 and DL-91) must await further study. Electrophoretically, they appear distinct from all other species with their nearest relative being C. aculabrum. Initial results indicate that morphologically they more closely resemble C. setosus. Based on morphology of the gonopods from two Male-I individuals, H. H. Hobbs, Jr. assigned the population in DL-91 to C. setosus. Although my examination of Male-I gonopods from DL-38 and DL-74 indicate that they have a C. setosus-type of morphology, voucher specimens need to be collected from those caves and examined under a microscope. (Toward that end, a Male-II from DL-38 is being held in captivity until it molts to Male-I condition.)

The conclusions that can be drawn from this study are summarized in Table 3. Cave crayfish occur in only a few caves in Oklahoma with four caves having persistent long- term use by individuals. Two other caves appear to be used intermittently, and a third cave could not be located. Of the four caves found to have persistent use, one is the only documented site for C. tartarus. The other three caves appear to have a second species initially identified as C. setosus. The electrophoretic studies that were conducted indicate that cave crayfish from these three caves are genetically unique from C. setosus in southern Missouri. Additional morphometric study is needed before a species determination can be made.

1. Cambarus tartarus should be listed as an endangered species on the basis of its morphologic distinctness and its rarity.

2. Cambarus aculabrum should be accepted as a distinct species, based on both its morphologic and genetic distinctness. It is an extremely rare species, known only from two caves, and it is dependent on water from the Ozark karst hydrologic system that is vulnerable to rapid pollution. Based on its extreme rarity and the vulnerability of the water system to pollution, this species should be listed as endangered.

3. Voucher Male-I specimens need to be collected from DL-38 and DL-74 in order to firmly establish the identity of cave crayfish populations from those caves and DL-91. If morphological differences are found between those populations as a group and C. setosus. they may merit designation as a distinct species, since genetically they are more closely aligned with C. aculabrum than with C. setosus.

4. Caves DL-49 and DL-64 should be revisited immediately following a rainy season that causes water levels to rise significantly. Cave crayfish appear more likely to be found during high water intervals. Table 3. Summary of current status of species determinations for cave crayfish (Cambarus spp.) in Oklahoma.

Cave Site Species determination based on additional Code No. Morphology Electrophoresis work needed

DL-91 1 C. se£Osus new species Conf1Illl morpho identification.

DL-38 2 C. setosus? new species Collect Male-I for morpho identification.

DL-74 12 C. setosus? new species Collect Male-I for morpho identification.

Spring Creek C. setosus? Find cave; collect collect Male-I for morpho & electro. identification.

DL-39 6 C. tartarus C. setosus List as endangered.

DL-49 10 * Continue to check cave for individuals.

DL-64 14 ** Continue to check cave for individuals.

*Tentatively assigned to C. tartarus based on proximity to DL-39. **Tentatively assigned to C. tartarus based on shared surface drainage with DL-39. 5. Caves DL-21 and OT-19 should be surveyed for cave crayfish during routine surveys for other cave species such as Myotis grisescens and Amblyopsis rosae. Along with DL-64 (see Recommendation 4), they are caves with records of A. rosae, so habitat for cave crayfish appears to exist in them. 6. Efforts should continue to locate the cave on Spring Creek in Mayes County and to identify the cave crayfish inhabiting it.

7. Hydrologic and pollution studies of the subterranean regions around caves DL- 74 and DL-39 need to be conducted. Both caves have substantial numbers of cave crayfish, which are vulnerable to changes in water quality and quantity. Fortunately, hydrologic and pollution studies are underway for caves DL-91 and DL-38 as part of recovery work with Amblyopsis rosae.

8. Protection efforts for C. tartarus in DL-39 should continue since this is the only cave in the world documented to have that species. Even if cave crayfish from DL-49 and DL-64 are found to be C. tartarus. the largest population remains in DL-39.

9. If the species in DL-38, DL-74 and DL-91 is determined to be a unique taxa, those caves would merit strong protection as well. Because DL-38 and DL-91 harbor the cavefish in addition to cave crayfish, protection of those caves would be expected to be forthcoming as part of recovery efforts for that species.

10. The electrophoretic studies of troglobites in the Jugicambarus group needs to be completed. In particular, C. zophonastes from Hell Creek cave and C. aculabrum from Logan cave in Arkansas need to be sampled. Although the two known sites for C. aculabrum are on surface drainages that flow into the Neosho River system, Bear Hollow Cave surface drainage flows into a tributary of the to the north in Missouri, while Logan Cave surface drainage is into the Illinois River far to the south. Thus, it is possible that the subsurface waters may no longer be connected between these caves. Because C. zophonastes is found far to the east of all other known species of Jugicambarus. it would be expected to show greater genetic distance from the other species. One C. setosus site in extreme southwestern Missouri, south of Neosho, has not been sampled electrophoretically. Because of its proximity to Oklahoma caves, it merits sampling to determine its genetic relatedness to the C. setosus-types in Oklahoma.

11. Cave crayfish in the Ozark Plateau have not been studied in any depth. In order to know protection needs for these species, we need to know the nature and degree of their dependency on caves as well as basic population parameters. Toward this end, a method of following individuals over time through mark-recapture or other techniques should be worked out and initiated. Southern Missouri caves that have relatively large populations of the more abundant species, C. setosus, probably is the best area to initiate this work. However, if electrophoretic studies continue, marking individuals from which claw samples are taken would allow a test of marking techniques concomitant an assessment of the impact of claw removal on individuals. Aley, T. 1989. Hydrology of Ozark cavefish caves in Oklahoma. Progress report #3 to The Nature Conservancy. Tulsa, Ok.

Black, J.H. 1971. Cave life of Oklahoma. Oklahoma Underground, Central Ok. Grotto, 4(12):2-53.

Creaser, E.P. and A.I. Ortenburger. 1933. The decapod of Oklahoma. Publications of the University of Oklahoma Biological Survey. 5(2):14-47.

Figg, D.E. and K.B. Lister. 1990. Status survey of the troglobitic crayfish Cambarus setosus in Missouri. Unpubl. report by the Missouri Dept. of Conservation. Jefferson City.

Gardner, J.E. 1986. Invertebrate fauna from Missouri caves and springs. Conservation Commission of the State of Missouri. Columbia. 72 pp.

Hobbs, H.H. Jr. 1976. (Astacidae) of North and Middle America. U.S. Environmental Protection Agency. Water pollution control research series 18050 ELD05/72. Cincinnati, Ohio.

Hobbs, H.H. Jr. and T.e. Barr, Jr. 1960. The origins and affinities of the troglobitic crayfishes of North America (, Astacidae). American Midland Naturalist. 64(1):12-33.

Hobbs, H. H. and M. S. Bedinger. 1964. A new troglobitic crayfish of the genus Cambarus (Decapoda, Astacidae) from Arkansas with a note on the range of Cambarus cryptodytes Hobbs. Proceedings of the Biological Society of Washington 77(3): 9-15.

Hobbs, H.H. Jr. and A.V. Brown. 1987. A new troglobitic crayfish from Northwestern Arkansas (Decapoda: ). Proceedings of the Biological Society of Washington. 100(4): 1040-1048.

Hobbs, H.H. Jr. and M.R. Cooper. 1972. A new troglobitic crayfish from Oklahoma (Decapoda: Astacidae). Proceedings of the Biological Society of Washington. 85(3):49-56.

Hobbs, H.H. Jr., H.H. Hobbs III, & M.A. Daniel. 1977. A review of the troglobitic decapod crustaceans of the Americas. Smithsonian contributions to zoology. No. 244. 183 pp. Koppelman, J.B. 1990. A biochemical genetic analysis of troglobitic crayfish (Cambarus spp.) in Missouri, Oklahoma, and Arkansas. Unpubl. report for the Missouri Dept. of Conservation, Oklahoma Natural Heritage Inventory, and Arkansas Game and Fish Commission. Missouri Dept. of Conservation. Columbia.

Looney, M. 1975. Cambarus tartarus: January River's blind crayfish. Okla. Underground 7(2):13-14.

Mayden, R. L. and F. B. Cross. 1983. Reevaluation of Oklahoma records of the southern cavefish. Southwestern Naturalist 28:471-473.

Williams, A.B. 1954. Speciation and distribution of the crayfishes of the Ozark plateaus and Ouachita provinces. The Univ. of Science Bulletin. 36(2):803-918.

REPORT PREPARED BY OKLAHOMA OKLAHOMA NATURAL HERITAGE INVENTORY

(/;O/Pi) Date 1

APPROVED BY OKLAHOMA DEPARTMENT OF WILDLIFE CONSERVATION

// #~ ~ ~~~ Harold E. Namminga r:7' Federal Aid/Research Coordinator Appendix I. Sites surveyed for crayfish in Oklahoma in 1989 and 1990. Results of field and historical record surveys are included. All caves that were located are reported to the nearest four sections (4 square miles). 13: including 8 female, 2 male-II, Oklahoma Natural Heritage 1 male-I Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH74)

W.L. Puckette, personal communication

Specimen 148793,U.S. National Museum, colI. C. Rudolph

Specimen 133842, U.S. National Museum, colI. H.H. Hobbs III

Specimen 132575, U.S. National Museum, colI. J. Black

Specimens in H. Hobbs colI. #122350-1, U.S. National Museum, colI. A. Blair

Site Description: A large solution cave in limestone. Crayfish have been documented from the main (larger) pool and from the stream that is crossed to get to the main pool. A stream access point near the entrance and a second, Comments: On 8 Mar 90, H.H. Hobbs, Jr. reconfirmed the identification of Specimen 133842 as C. setosus. The specimens collected in 1951 were from "cave between Spavinaw and Jay" which is presumed to be Twin Cave.

Electrophoretic analysis by Koppelman (1990) suggests that this is a new species most closely aligned with C. aculabrum. ~7- '-=:-/\ '/' /.;....-l v'r-

/' / / / / - A 4 seen: includes 1 female, 1 Oklahoma Natural Heritage male-II Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F90MEH03)

Aley (1989)

R. Hamilton, The Nature Conservancy, personal communication

4 seen: includes 1 female, 1 Oklahoma Natural Heritage male-II, 1 male-I Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH75)

Specimen 148794, U.S. National Museum, colI. C. Rudolph

Site Description: A solution cave in limestone. Crayfish were seen during this survey only in the single pool in the cave. A stream is present, but little of it can be accessed for survey. the cave was a maternity site for Myotis grisescens (gray bats) which brought nutrients into the cave through guano. Water pollution from external sources could be a threat in the future. Hydrology of the cave could be altered by rises in the level of Grand Lake.

Comments: H.H. Hobbs, Jr. tentatively assigned specimen 148794 to C. setosus, though previously (Hobbs et al 1977) it was assigned to C. tartarus with no specimens having been examined. A male-II was taken alive by Mehlhop- Cifelli on 26 May 90 to hold until it molts to male-I and can be identified morphologically.

Electrophoretic analysis by Koppleman (1990) suggests that this is a new species most closely aligned with C. aculabrum. -9<~':)-~=--~3

, ' \ Dates Surveyed & Source of Data: 27 Feb 51: 1 immature female was collected by AP. Blair (H.H. Hobbs, Jr. #5-2150-1, U.S. National Museum). No records of a cave or troglobite have been made since that time. Contact was made October 89 with four long-term residents of the south slope of Spring Creek. None of them had heard of a cave being present. No cave crayfish were found in a field search along the slopes of the creek 26 October 1989.

Site Description: Along Spring Creek, limestone occurs intermittently, mostly on the North side.

Comments: J.H. Black (personal communication) suggested that the source could have been a well-known spring 0.5 mi. E. of Locust Grove (Site 17). The spring has been closed except for a pipe from which the water flows. If a cave crayfish occurred in the spring, it would have been ejected from more suitable habitat underground and upstream.

H.H.Hobbs, Jr. has tentatively assigned this to Cambarus setosus. but a Male-I needs to be collected to confirm the identification. .-.-/) • ,j

Mayes County I Oklahoma Historic Site: no cave located quad map: Locust Grove T19NR20E Sec. 9 Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and others (F89MEH79)

1 male-II, held until molted to Specimen 131951, U.S. National male-I Museum, colI. J.H. Black. (molted 7 May 1971)

Specimen 131411,U.S.National Museum, colI. J.H. Black

Specimen 132754,U.S. National Museum, colI. J.H. Black

Site Description: A long solution cave in limestone. A stream runs the length of the cave which does not occur in a resurgence area.

Comments: This is the type locality for this species. Brown (1986) indicated he has seen the species in the cave, but gave no data. .-,,-", __ 0.... 26_

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.-:-r-=- .

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Delaware County, Oklahoma ;\ cave DL-39, Site 6 )'\ , / quad map: Colcord T21NR24E Sees. 1,2,11,12 Dates Surveyed and Source of Data: 10/28/89: 17 seen, including 7 females, 3 Male- II, 1 Male-I, Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli & William Puckette (F89MEH82); Puckette knew of crayfish in the cave prior to 1989 (personal communication); reported by Black (1971)

Site Description: A long solution cave in limestone. Six small pools were found in 1989 survey, all of which had cave crayfish. Loose rock slabs in the crawl spaces mandate extreme caution by humans visiting the cave.

Comments: Hobbs et. al. (1977) tentatively assigned the species to C. tartarus without having seen specimens. On the basis of visual observation of male-I gonopods and robustness of the chelae, Mehlhop-Cifelli would not assign it to C. tartarus. .. - r,--= )~ Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F90MEHOl)

Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH80)

W.L. Puckette and J. Stewart (personal communication)

Reported in the 3 caves (sites 8-10) with no detail in article

Site Description: A series of small mud bottomed solution caves running east-west along a limestone cliff. The easternmost cave, site 7, is a small sump where no water was seen. Site 8 has about 2m of visual access to a stream. Site 9 has a small sump within it in which Amblyopsis rosae were seen in the past. There is also a small stream. Site 10 has a small pool near the entrance which was dry 10/23/89, but wet 5/26/90. Also, it has a muddy crawl area that is usually with water, but was dry 10/23/89. These caves flooded to a height of more than 4 feet during spring 1990 rains, but waters appear to have receded quickly. Threats: Water pollution could be a threat in the future, particularly from increasing pig and chicken farming.

Comments: Based on proximity to cave DL-39, the species may be Cambarus tartarus. Puckette and Mehlhop-Cifelli believe that cave crayfish use site 10 and probably sites 8 and 9 when water levels are sufficiently high in the caves. The caves may act as a significant nutrient source for the crayfish and other subterranean species when mud washes into the caves during flooding. There is some use of the caves by bats, including sign of small groups of Myotis grisescens. indicating that bat guano may be a significant source of nutrients as well. o. o ,/ / -- . --6--- - L - ~_io- _L_ '~ ~ ,

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Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH84)

Black (1971), W. L. Puckette (personal communication)

Site Description: A limestone solution cave just above an upper resurgence area to the underground stream. Troglobitic species, therefore, would be expected upstream rather than downstream from the cave. There is a moderately swift flowing stream only 3m length of which can be accessed. A moderate- sized pool with stream flow through it was very low 10/28/89, but about 3 feet and rising during rain 5/26/90. Apparently, water levels in it rise and fall rapidly. A second very small pool was dry 10/28/89 and held little water 5/26/90. Comments: High water level fluctuations and current flows may not be conducive to frequent use by cave crayfish.

Hobbs et al (1977) tentatively assigned the species to C. tartarus without examination of specimens. Because this cave is in the same major surface drainage as DL-39, that tentative assignment is appropriate.

Dates Surveyed and Source of Data: W.L. Puckette has surveyed the cave 3 times and not seen a cave crayfish. Mary Looney (1975) mentioned that cave crayfish had been reported from the cave, but no records have been located to substantiate it.

Site Description: A narrow solution cave in limestone. A perennial stream runs along the length of the cave. Water flushes through the cave without pool formation.

Comments: According to W.L. Puckette, the cave probably is not a suitable long term habitat for troglobites because they would be flushed from the cave during high water periods.

If cave crayfish occur in this cave, they may be C. tartarus based on proximity to DL-39. o ~_ • "036 --0----- .. ~ ·~·.II<> ~ ~.o

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Delaware County, Oklahoma Cave DL-19 ~- quad map: Colcord / T21NR24E Sees. 2,3,10,11 USGS Quadrangle Map: Lake Eucha East

, Site Location to Nearest 4 Sections: T21NR23E

Dates Surveyed and Source of Data: None seen by Black (1971). None seen by W.L. Puckette in winter visits to cave from 1980-1989. 1983: none seen by Puckette and J. Stewart (personal communication). 5 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and William Puckette (F89MEH76)

Site Description: A solution cave in limestone of moderate size. A stream meanders through the cave and permits access along approximately 50 m.

Comments: A long history of biological survey of this cave has not revealed any evidence of cave crayfish presence. Delaware County, Oklahoma cave DL-2l, Site 3 quad map: Lake Eueha East T21NR23E Sees. 7,8,17,18 ·Dates Surveyed and Source of Data: 5 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH77)

Site Description: A shallow solution cave in limestone. Water covers the cave from the entrance to as far back as can be accessed, which is just beyond the light zone. Water covers the bottom of the cave from the entrance to as far back as can be accessed. ~- -- -~

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,--~ Dates Surveyed and Source of Data: 5 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH78)

Site Description: A shallow solution cave in limestone with no apparent access beyond the dim light zone. Water fills the cave bottom.

Dates Surveyed and Source of Data: 26 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH81)

Site Description: A moderate size cave in limestone. There is a small stream that is crossed to get to a moderate pool.

Threats: Flocculent on the pool surface indicates water pollution, probably from the cave owners house above the cave.

Comments: This cave probably is in the same major drainage (Sycamore Creek) where Amblyopsis rosae was exposed during a construction project. It is the only known aquatic cave of noteworthy size in the drainage. S. Bozeman (Central Oklahoma Grotto, personal communication) knows of no records of cave crayfish from this cave, nor does W.L. Puckette. I :: 0 0 C -',7 ~l , Delaware County, Oklahoma Cave DL-84, Site 11 quad map: Sycamore T23NR24E Sees. 11,12,13,14 Dates Surveyed and Source of Data: 28 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH83)

Site Description: A shallow solution cave in limestone with several entrances. There is little dark zone accessible in the cave. /~~.~

$46 _-----. '0 v--:-.., r, o : -Dates Sunreyed and Source of Data: 28 Oct 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH85)

Site Description: A small solution cave in limestone. A dry gravel stream bed was visible, but could not be accessed without digging.

Dates Surveyed and Source of Data: None recorded as being seen during surveys for Amblyopsis rosae in 1954, 1966 and 1980-83 (Mayden & Cross 1983). 7 Dec 89: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli and W.L. Puckette (F89MEH84)

Site Description: A solution cave in limestone. A rock and gravel stream runs through the cave and goes slightly under ground just inside the cave entrance. Access is easy the first 100m or more into the cave, but much of the stream flows beneath a rock shelf.

Threats: Water pollution is a potential threat. An impoundment below the cave does not appear to threaten water back up into the cave. However, if back up did occur, predatory fish could enter the cave.

Comments: There is no evidence of cave crayfish from this cave, although the habitat appears suitable. Any troglobites in this cave may be getting flushed by the stream from more populous and inaccessible reaches upstream. Ottawa County, Oklahoma cave OT-19, Site 16 < c; quad map: Peoria .-'"""1- ,.,.,"1o"m"1 ....t:•" ~--. __ ·Dates Surveyed and Source of Data: 26 May 90: Oklahoma Natural Heritage Inventory field survey by P. Mehlhop-Cifelli (F90MEH04)

Site Description: A spring in a roadside park 0.5 mi. E. of Locust Grove. The spring has been capped. A pipe has been inserted to let the spring water run freely into a small drainage.

Comments: J.H. Black (personal communication) suggested that this might be the actual locality for the Spring Creek specimen. If cavefish occur in this system, they could be observed only if flushed from the spring through the pIpe. '( - . ---~-~._.-- ... ::;) _---':--~-~~~:...• ., ~;.::.-:;~~~

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Mayes County, Oklahoma Spring, Site 17 quad map: Loeust Grove T20NR20E Sees. 14,15,22,23 1/\ Appendix II. Report from Dr. Jeffrey Koppelman of the Missouri Department of Conservation on results of electrophoretic analyses of tissue proteins from cave crayfish in the Ozark Plateau. A Biochemical Genetic Analysis of Troglobitic Crayfish

Jeffrey B. Koppelman Fisheries Research Biologist Background

This research study was a preliminary investigation of the feasibility of using electrophoretic techniques such as those currently in use in the Fish Genetics Laboratory to examine systematics of cave crayfish (Decapoda: Cambarus §QQ.) in the Ozarks region. Five species of troglobitic Cambarus have been reported from this region: C. aculabrum, C. hubrichti,

C. setosus, and C. tartarus and C. zophonastes. Morphological identification of these crayfish is difficult because Form I males are needed, and these may not be available in the small samples that can be obtained at a specific site at anyone time. Biochemical genetic analysis has the potential advantage of providing a large amount of information from a few individuals from different populations since there is little gene exchange between these populations. Past investigations have revealed that epigean crayfish species have relatively low levels of heterozygosity (Nemeth and Tracey 1979; Brown 1981). The study objective was to determine whether electrophoresis can be used to distinguish between different species and to assess intraspecific genetic variability.

Methods

Because individual cave populations are relatively small, it was decided that chela muscle would be used for isozymic analysis to avoid population reductions. Although population genetic variation estimates are strongly enhanced by increased sample sizes, greater strength is gained through increased numbers of gene loci. A total of 48 individuals were captured from 20 different cave sites in Missouri, Oklahoma, and Arkansas during fall and winter, 1989, representing four of the five species in the tri-state region (Table 1). Specimens of

C. zoohonastes, known only from a single site in Arkansas, were not collected. The left chela was removed from each collected crayfish, which was then released.Cave crayfish missing a left chela were observed in subsequent visits to some sites, indicating that they survived this

method of tissue sampling.

All chelae were held on dry ice and placed in an ultra-low freezer (-80°C). To process

the tissues for analysis, each whole claw, including the chitinous exoskeleton, was crushed,

mixed with a buffer, homogenized, and centrifuged for 30 minutes at 12.000 x g. Samples were

electrophoresed and subjected to histochemical staining procedures (Philipp and Whitt 1979;

Koppelman and Philipp 1986). The resulting phenotypic banding patterns were used to directly

infer genotype, and the resultant individual allelic information was statistically analyzed using

the BIOSYS-1 program of Swofford and Selander (1981).

Results

Approximately 30 specific enzyme and general protein staining techniques were used to

reveal gene products present in cave crayfish chelae muscle tissue, and 20 gene loci could be

reliably scored using these methods. Thirteen loci were polymorphic (Table 2); the maximum

number of alleles observed at a single locus was three (Table 2). However, only four

populations had any heterozygosity: Fauna Cave, Sequiota Cave, Smallin's Cave. and

Oklahoma Site 1. The actual amount of heterozygosity may have been greater, since an

average of only 2.4 individuals were sampled per site. Of these four populations, Sequiota

Cave had the greatest amount of direct-count heterozygosity (Table 3).

5 Contingency chi-square analyses confirm (P<1 0. ) that these samples do not represent

one large, randomly breeding population. Differentiation levels for the cave crayfish populations were very high for all polymorphic loci (mean FoT=0.963). In term's of Nei's gene diversity

analysis, 3.7% of the total gene diversity is included in the within-deme component; the among- deme diversity represents 96.3% of the total. Examining C. setosus together, mean FoT=0.924. Rogers distance measure clusters three main groups of populations at or above the

0.249 level (Figure 1). All three Q. hubrichti populations are very similar (0=0.00) and they are very different from the other populations (0=0.423). C. aculabrum and Sites 1, 2 and 12

comprise a second cluster, although there is a moderate amount of differentiation (0=0.201)

between C. aculabrum and these three Oklahoma sites. Sequiota, Smallin's, Fauna, and

Fulbright caves were different from each other but they were clustered together separate from

the remaining C. setosus populations and the single C. tartarus sample site, all of which were

virtually identical (0=0.00).

Discussion

Several loci in addition to those reported could possibly be resolved for inclusion in

future analyses. Thirteen polymorphic loci out of twenty is average among congeners. The low

average number of samples per collection site does not allow for accurate representation of

heterozygosity within each population. Under these conditions, it is unusual to see 15%

polymorphic loci in population samples as small as those for Sequiota and Smallin's caves. It is

even more interesting that these two caves are proximal in Greene County, Missouri.

It was expected that contingency chi-square analysis would confirm that all of these

populations were not one large, randomly breeding population due to already confirmed multiple

taxa. It is also not that unusual to observe the level of differential fixation found for a species

complex represented by such small sample sizes. Fixation within C. setosus was similarly high,

probably resulting from small sample sizes.

C. hubrichti represents the only troglobitic species in the subgenus Erebicambarus, and

it was correspondingly the most genetically distinct species evaluated. The most significant finding was different clustering of three groups of C. setosus, and the genetic relatedness of one of these groups to the C. tartarus population sample. C. aculabrum and Oklahoma C. setosus clustered together but at a distance level great enough to indicate that the two groups are genetically distinct. The remaining C. setosus were divided with a notable east to west split.

Fantastic Caverns, however, was more proximal to the east group but clustered with the west group. The presence of variation in all of the eastern populations and an absence of variation in the western group, including Fantastic Caverns, accounted for the pairwise distance comparison separation. However, failing to detect variation in the western group, a likely outcome of small sample size, would not necessarily account for the three variable populations clustering together. Therefore, there is reason to conclude that there is some level of

·uniqueness· in populations of C. setosus from the Greene County, Missouri, area. There is likewise something unique about Oklahoma C. setosus populations, producing a phenetic relationship intermediate between the western Missouri conspecifics and C. aculabrum.

Recommendations

There is ample evidence that the use of starch gel electrophoresis can provide the necessary resolution to examine intra- and interspecific genetic variation in troglobitic crayfish. provided that either the number of caves or the number of individuals sampled from each cave are increased. While heterozygosity is undoubtedly underestimated to some degree with small population sample sizes, the phenomenon of low heterozygosity among decapod crustacea has been well documented (Nemeth and Tracey 1979; Brown 1981). Therefore, variablility levels may be very low in cave crayfish populations. The fact that most geographically similar populations also clustered closely supports continued electrophoresis efforts. Future research should be designed to accomplish two objectives. First, an accurate genetic evaluation of Ozarks representatives of both subgenera involved, Erebicambarus and

Jugicambarus, is necessary to put into perspective the amount of differentiation observed

between C. hubrichti and the other troglobitic species, which belong to the sUbgenus

Jugicambarus. Analysis of additional C. hubrichti, as well as analysis of two epigean

consubgeners, C. maculatus and C. hubbsi, would be required. C. zophonastes, known only

from one location in Stone County, Arkansas, C. aculabrum from a second site in Benton

County, Arkansas, additional C. tartartus from the only known site in Delaware County,

Oklahoma, and additional C. setosus from both the Springfield, Missouri, and southwestem

Missouri areas should be genetically evaluated.

The second objective should be to determine the extent of genetic differences between

cave crayfish from the central region of Delaware County, Oklahoma, including Sites 1, 2 and

12, and other C. setosus in adjacent regions . ..Q.. aculabrum from both known sites in Arkansas

should be sampled for comparison. It is possible that there is a separate taxon of cave crayfish

distributed in this range in Oklahoma, or a continuous distribution of allele frequencies may exist

between these taxa, indicating subspecific status. Brown, K. 1981. Low genetic variability and high similarities in the grayfish genera Cambarus

and . The American Midland Naturalist 105(2):225-232.

Koppelman, J.B., and D.P. Philipp. 1986. Genetic applications in muskellunge management.

American Fisheries Society Special Publication 15:111-121.

Nemeth, S.T., and M.L Tracey. 1979. Allozyme ~ariability and relatedness in six crayfish

species. The Journal of Heredity 70:37-43.

Philipp, D.P., W.F. Childers, and G.S. Whitt. 1979. Evolution of patterns of differential gene

expression: a comparison of the temporal and spatial patterns of isozyme locus

expression in two closely related fish species (northern largemouth bass, Micropterus

salmoides salmoides, and smallmouth bass, Micropterus dolomieui). Journal of

Experimental Zoology 175:283-296.

Rogers, J. S. 1972. Measures of genetic similarity and genetic distance. Studies in Genetics,

University of Texas Publication 7213:145-153.

Swofford, p. L, and R. B. Seland. 1981. BIOSYS-1: a FORTRAN program for the

comprehensive analysis of electrophoretic data in population genetics and systematics.

Journal of Heredity 72:281-283. Table 1. Cave crayfish (Cambarus ~.) populations, county, state, species and the number of Individuals sampled.-

Population County State Species N

1. Lewis Cave Carter Missouri hubrichti 2 2. Medlock Cave Shannon Missouri hubrichti 1 3. Deadman Cave Oregon Missouri hubrichti 2 4. Bear Hollow Cave Benton Arkansas aculabrum 2 5. Fantastic Caverns Greene Missouri setosus 2 6. Fulbright Spring Cave Greene Missouri setosus 1 7. Fauna Cave Greene Missouri setosus 1 8. Sequiota Cave Greene Missouri setosus 3 9. Smallin's Cave Christian Missouri setosus 3 10. Coolbrook Cave Jasper Missouri setosus 1 11. Kellhofer's Cave Jasper Missouri setosus 5 12. Wilson's Cave Jasper Missouri setosus 1 13. Elm Spring Newton Missouri setosus 1 14. Hearrell Spring Newton Missouri setosus 1 15. Hayes Spring Cave Stone Missouri setosus 2 16. Carrico Cave Dade Missouri setosus 1 17. Site 1 Delaware Oklahoma setosus 7 18. Site 2 Delaware Oklahoma setosus 2 19. Site 6 Delaware Oklahoma tartarus 6 20. Site 12 Delaware Oklahoma setosus 4

- Population numbers correspond to those in Table 2. Table 2. Allele frequencies sl each polymorphic locus surveyed. Populallon number. correspond 10 Ihose In Table 1.

Populallon

3 5 6 7 8 9 10 11 12 13 14 --Locus 1 2 4 15 16 17 18 19 20

AAT-M (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 .000 .000 .000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 1.000 1.000 1.000 .000 1.000 .000 .500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 3 .000 .000 .000 .000 .000 1.000 .500 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000

AAT-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 .000 .000 .000 .000 .000 1.000 1.000 1.000 .333 .000 .000 .000 .000 .000 .000 _000 .000 .000 .000 .000 2 1.000 1.000 1.000 1.000 1.000 .000 .000 .000 .667 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

AOA-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

AOK-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

AID-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

FDP-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 .000 .000 .000 .000 .000 .000 .000 .667 .333 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .333 .667 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

GAP-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 Table 2. (Continued)

GPI-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 .000 .000 1 1.000 1.000 1.000 .000 .000 .000 1.000 .333 1.000 .000 .000 .000 .000 .000 .000 .000 .071 .000 2 .000 .000 .000 1.000 1.000 1.000 .000 .667 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .929 1.000 1.000 1.000

IOH-A 4' (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 1 1.000 1.000 1.000 .000 .000 .000 .000 .333 .167 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 .000 .000 .000 .000 1.000 1.000 1.000 .667 .833 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .000 .000 1.000 .000 3 .000 .000 .000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 1.000 1.000 .000 1.000

IOH·B (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 .000 .000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .000 .000 1.000 1.000 3 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 1.000 1.000 .000

LOH-A 6 4 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 1.000 1.000 1.000 1.000 1.000 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

MOH-A 6 4 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 1.000 1.000 1.000 1.000 1 .000 .000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .000 .000 2 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000

MOH-B 6 4 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 1.000 1.000 1.000 1.000 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

MEN-A 7 2 6 4 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 1.000 1.000 .000 .000 1.000 .000 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .000 1.000 2 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 1.000

PGO-A 7 6 4 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 2 .000 .000 .000 .000 1 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 1.000 .000 2 .000 .000 .000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 .000 1.000 3 .000 .000 .000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 Table 2. (Continued)

PGM-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 .000 .000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

PRO-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4' 1 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

PRO-8 (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 .000 .000 .000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 1.000 1.000 1.000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

PRO-C (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 .000 .000 .000 .000 .000 1.000 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 3 .000 .000 .000 1.000 1.000 .000 .000 .000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

SOD-A (N) 2 1 2 2 2 1 1 3 3 1 5 1 1 1 2 1 7 2 6 4 1 .000 .000 .000 .000 .000 .000 .000' 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 2 .000 .000 .000 1.000 1.000 1.000 1.000 .000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 3 1.000 1.000 1.000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 Genetic variability at 20 gene loci In cave crayfish. A locus Is considered polymorphic If more than one allele was detected. Mean heterozygosity is determined by direct count method.

Sample % Loci Mean Size Polymorphic Heterozygosity

1. Lewis Cave 0.0 0.000 2. Medlock Cave 0.0 0.000 3. Deadman Cave 0.0 0.000 4. Bear Hollow Cave 0.0 0.000 5. Fantastic Caverns 0.0 0.000 6.Fulbright Spring Cave 0.0 0.000 7. Fauna Cave 5.0 0.050 8. Sequiota Cave 15.0 0.033 9. Smallin's Cave 15.0 0.017 .10. Coolbrook Cave 0.0 0.000 11. Kellhofer's Cave 0.0 0.000 12. Wilson's Cave 0.0 0.000 13. Elm Spring 0.0 0.000 14. Hearrell Spring 0.0 0.000 15. Hayes Spring Cave 0.0 0.000 16. Carrico Cave 0.0 0.000 17. Site 1 5.0 0.007 18. Site 2 0.0 0.000 19. Site 6 0.0 0.000 20.Site 12 0.0 0.000 Unweighted pair group cluster analysis of genetic distance (0) values (Rogers 1972). based on all loci surveyed.

+----+----+----+----+----+----+----+----+----+----+----+----+----+--- -+---- +---- + .48 .42 .36 .30 .24 .18 .12 .06 .00